CN116622806A - Method for improving RNA virus detection rate - Google Patents
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Abstract
The application relates to a method for obtaining viral RNA, comprising the following steps: removing host rRNA in the first nucleotide set to obtain a second nucleotide set; wherein the first set of nucleotides includes host DNA, host rRNA, and pathogen RNA; the second set of nucleotides includes pathogen RNA; and purifying pathogen RNA in the second set of nucleotides. The technical scheme of the application has simple flow, high detection sensitivity and high detection rate of RNA pathogens, and has very important reference significance for solving the problems of low viral load of clinical samples and the like.
Description
Technical Field
The present application relates to pathogen detection methods, and in particular to a method for increasing the detection rate of RNA viruses.
Background
RNA viruses play a significant role in the occurrence and development of infectious diseases, and the identification of RNA viruses and transcriptionally active microorganisms in a sample has important clinical significance in assisting in the diagnosis of infectious diseases.
The traditional methods for identifying viruses in the clinical microorganism laboratory mainly comprise separation culture, immunofluorescence detection, PCR molecular detection and the like, and have obvious limitations. The macro transcriptome sequencing of the viruses can comprehensively and unbiased detect RNA viruses in samples, can timely discover novel pathogens and mutations thereof which cannot be detected by the traditional method, and provides more accurate reference for assisting clinical decision. Compared with the traditional virus identification method, the existing high-throughput sequencing technology has the advantages of short period, low cost, general technical requirements for operators and the like, and is accepted by more and more clinical departments.
In the research field of RNA virus macro transcriptome, the nucleic acid content of clinical sample host is extremely high and can reach 10 6 The individual/mL level interferes with accurate detection of RNA virus. The conventional method for removing host nucleic acid is to digest host DNA by chemical means or differential lysis of host cells and then use nuclease. However, RNA viruses exist in the sample in the form of free pathogens or nucleic acids, and the viruses have a simple structure relative to other microorganisms, are easily cleaved to release nucleic acids, and the released nucleic acids are further degraded by nucleases of the host and other microorganisms. The host nucleic acid removal method in the prior art also loses RNA virus while removing host nucleic acid, and causes false negative of detection results; in addition, the sequencing result of RNA viruses often shows a high human proportion, which affects the detection rate of RNA viruses. Thus, the detection of RNA viruses in clinical samples by existing sequencing methods generally does not involve removal of host nucleic acids.
Disclosure of Invention
Aiming at the technical problems in the prior art, the application provides a method for obtaining viral RNA, which comprises the following steps: removing host rRNA in the first nucleotide set to obtain a second nucleotide set; wherein the first set of nucleotides includes host DNA, host rRNA, and pathogen RNA; the second set of nucleotides includes pathogen RNA; and purifying pathogen RNA in the second set of nucleotides.
The method as described above, further comprising: removing DNA in the first nucleotide set to obtain a first RNA set; the first RNA set includes: host rRNA and pathogen RNA; enriching the first RNA set in the total nucleotides of the sample to obtain an enriched first RNA set; removing host rRNA in the enriched first RNA set to obtain a second RNA set, wherein the second RNA set comprises pathogen RNA; and purifying pathogen RNAs in the second RNA collection.
The method as described above, wherein the second set of nucleotides comprises a second set of RNAs.
The method of claim 2 above, wherein the method of enriching the first set of RNAs comprises: the first RNA collection is enriched using first magnetic beads that are used in an amount of 1.6-2.0 times the sample volume, preferably 1.8 times.
The method as described above, further comprising: adding to the sample an rRNA probe configured to specifically bind to the host rRNA in the first nucleotide set or the first RNA set without binding to pathogen RNA; and removing complexes of the rRNA probes that bind to the host rRNA from the sample.
The method as described above, further comprising: RNase is added to the sample, which digests host rRNA bound to the rRNA probe, but does not digest pathogen RNA.
The method as described above, the method of purifying the pathogen RNA comprising: enriching the residual DNA and the residual RNA except for pathogen RNA in the second nucleotide set or the second RNA set using a second magnetic bead; the amount of the magnetic beads used is 2.0 times to 2.4 times, preferably 2.2 times, the sample volume.
A method as described above, wherein the method of enriching pathogen RNA in a sample comprises: the pathogen RNA is enriched using a third magnetic bead in an amount of 2.0-2.4 times the sample volume, preferably 2.2 times.
The method as described above, wherein the sample is alveolar lavage fluid, cerebrospinal fluid, blood, urine, stool, respiratory tract sample, hydrothorax, ascites, pericardial fluid, vomit, abscess tissue.
A method as described above, wherein the pathogen is one or more of the following RNA viruses: human orthopneumovirus, rhinovirus A, human metapneumovirus (hMPV), GBV-C, human respiratory virus 3, human parainfluenza virus type 2, influenza A virus, hepatitis A virus, rotavirus, measles virus, AIDS virus, encephalitis B virus, influenza B virus, rhinovirus, polio virus, coxsackie virus, dengue virus, rotavirus, SARS virus, MERS virus, ebola virus, marburg virus, phage, novel coronavirus (COVID-19).
A method of increasing the detection rate of RNA viruses in a sample, comprising: extracting sample nucleotides to obtain a first nucleotide set; wherein the first set of nucleotides comprises: host DNA, host rRNA, and pathogen RNA; purifying pathogenic RNA using the method of obtaining viral RNA according to any one of claims 1-10; reverse transcribing the pathogen RNA to obtain double-stranded pathogen cDNA; constructing the pathogen cDNA library; sequencing the cDNA library.
The method as described above, further comprising: purifying the cDNA.
Purifying the cDNA by electrophoresis, and/or adding third magnetic beads to purify the cDNA, as described above; the amount of the third magnetic beads used is 2.0 times to 2.4 times, preferably 2.2 times, the sample volume.
In the method described above, the cDNA library is 300-700bp in length of double-stranded pathogen cDNA.
A kit for purifying viral RNA from total nucleotides in a sample, comprising: a dnase configured to remove DNA in the first set of nucleotides to obtain a first set of RNAs; the first RNA set includes: host rRNA and pathogen RNA; or it is configured to remove DNA from the second set of nucleotides or the second set of RNAs; an rRNA probe configured to bind to host rRNA in the first nucleotide set or first RNA set, but not pathogen RNA; rnase configured to remove host rRNA bound to rRNA probes without removing pathogen RNA; RNA magnetic beads configured to enrich the first RNA pool and/or reverse enrich for pathogenic RNA after removal of host rRNA; the RNA magnetic beads comprise: the first magnetic beads and/or the second magnetic beads.
A kit for increasing the detection rate of RNA viruses in a sample, comprising: a dnase configured to remove DNA in the first set of nucleotides to obtain a first set of RNAs; the first RNA set includes: host rRNA and pathogen RNA; or it is configured to remove DNA from the second set of nucleotides or the second set of RNAs; an rRNA probe configured to bind to host rRNA in the first nucleotide set or first RNA set, but not pathogen RNA; rnase configured to remove host rRNA bound to rRNA probes without removing pathogen RNA; RNA magnetic beads configured to enrich the first RNA pool and/or reverse enrich for pathogenic RNA after removal of host rRNA; the RNA magnetic beads comprise: the first magnetic beads and/or the second magnetic beads.
Use of a method for obtaining viral RNA as defined in any of the preceding claims, or a method for increasing the detection rate of RNA viruses in a sample as defined above, for detecting an RNA pathogen in a sample.
Compared with the existing detection method, the application has the advantages that:
the method is suitable for, but not limited to, macro-transcriptome detection of samples such as human and animal cerebrospinal fluid, blood, alveolar lavage fluid, abscess tissue and the like;
the optimized host nucleic acid removal flow has no step of cracking host cells, and the degradation of RNA viruses is reduced to the greatest extent; the digestion step of the specific system further reduces host residues in the RNA used for pathogen identification, and the human proportion in the sequencing result is significantly reduced. Improves the detection sensitivity and is beneficial to the detection of RNA viruses.
Successfully connecting the rRNA removal step with the library construction process to obtain a standard library which can be used for on-machine sequencing, and successfully identifying the known pathogen; a specific library preparation flow containing human rRNA removal is constructed through flow detailed parameter optimization; the human source proportion of the sample in the sequencing result is extremely reduced, the effective microorganism reading length number and the pathogen detection reading length number are obviously improved.
The application adopts a reverse enrichment method to obtain the nucleic acid of the target pathogen, effectively improves the sensitivity of RNA virus macro transcriptome detection, and has very important reference significance for solving the problems of low viral load of clinical samples and the like.
Drawings
Preferred embodiments of the present application will be described in further detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a schematic flow chart after optimization for improving RNA virus detection according to one embodiment of the present application;
FIG. 2 is a plot of the proportion of nucleotides of human origin in the sequencing result after removal of sample DNA according to one embodiment of the application;
FIG. 3 is a graph showing the proportion of microbial nucleotides in the sequencing result after removal of sample DNA according to one embodiment of the present application;
FIG. 4 shows the proportion of human nucleotides in the sequencing result after removal of rRNA from a sample according to one embodiment of the present application; and
FIG. 5 shows the proportion of human nucleotides in the sequencing results after removal of rRNA from a sample according to one embodiment of the present application.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more apparent, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
In the following detailed description, reference is made to the accompanying drawings, which form a part hereof, and in which is shown by way of illustration specific embodiments of the application. In the drawings, like reference numerals describe substantially similar components throughout the different views. Various specific embodiments of the application are described in sufficient detail below to enable those skilled in the art to practice the teachings of the application. It is to be understood that other embodiments may be utilized or structural, logical, or electrical changes may be made to embodiments of the present application.
The terms used in the present application have the following meanings:
as used herein, "rRNA" refers to ribosomal RNA, which is the type of RNA having the greatest intracellular content, and also the type of RNA having the greatest relative molecular mass among 3 types of RNA (tRNA, mRNA, rRNA), and rRNA accounts for about 82% of the total RNA. In the present application, rRNA is rRNA of a pathogen host in a sample, which is a major component of host RNA. In some embodiments, the rRNA, the human RNA, and the human RNA are all host RNAs.
As used herein, "sample" refers to a sample to be tested obtained from a host that may be infected with a pathogen. Which contain host nucleotides and possibly pathogen nucleotides. In some embodiments, the sample may be alveolar lavage, cerebrospinal fluid, blood, urine, stool, respiratory tract samples, hydrothorax, ascites, pericardial fluid, vomit, abscess tissue, and the like. Among them, respiratory tract samples include, but are not limited to, nasal swabs, pharyngeal swabs, sputum, and the like. In some embodiments, the host may be a human, a non-human animal, or the like. Further, the non-human may be a mammal, such as a pet (cat, dog, netherlands pig, etc.), a mouse, rat, etc., or a primate (cynomolgus monkey, chimpanzee, etc.).
As used herein, "pathogen" refers to a pathogen that may infect a host. In some embodiments, the pathogen is an RNA pathogen. In some embodiments, pathogens include, but are not limited to: human orthopneumovirus, rhinovirus A, human metapneumovirus (hMPV), GBV-C, human respiratory virus 3, human parainfluenza virus type 2, influenza A virus, hepatitis A virus, rotavirus, measles virus, AIDS virus, encephalitis B virus, influenza B virus, rhinovirus, polio virus, coxsackie virus, dengue virus, rotavirus, SARS virus, MERS virus, ebola virus, marburg virus, phage, novel coronavirus (COVID-19), and the like.
The term "reference" as used herein refers to a pathogen in a sample that is to be detected, either as a pathogen in an infected positive sample or as a pathogen that has been artificially added to a negative sample for the purpose of performing the test.
As used herein, "optimized procedure", "post-optimized procedure" refers to the process of extracting the sample nucleotide prior to detecting RNA virus in the sample, removing DNA and rRNA therefrom, and then purifying, reverse transcribing, sequencing the remaining RNA to determine pathogen species, etc. The corresponding "control procedure" is to directly reverse transcribe and sequence the nucleotides without removing DNA and rRNA after extracting the nucleotides of the sample before detecting RNA viruses in the sample.
As used herein, the term "on-machine" or "off-machine" refers to an instrument for pathogen RNA detection. In some embodiments, the machine is referred to as a sequencer. In some embodiments, the method comprises sequencing and base reading the prepared pathogen cDNA library. In some embodiments, off-machine refers to a sequence file generated from sequencing and base reads obtained on-machine that can be used in bioinformatic analysis to obtain pathogen detection results.
As used herein, "first set of nucleotides" refers to the total sample nucleotides. In the present application, the first set of nucleotides is a set of nucleotides extracted in a sample. In some embodiments, the sample comprises nucleotides of the host and pathogen; the first set of nucleotides includes, but is not limited to, host DNA, host rRNA, host tRNA, host mRNA, pathogen RNA.
As used herein, the term "second nucleotide set" refers to a set of nucleotides from which host rRNA is removed based on the first nucleotide set, and which is selectively removed after host DNA. In some embodiments, the second set of nucleotides includes, but is not limited to, pathogen RNA, host rRNA, host tRNA, host mRNA. In some embodiments, host DNA is also included in the second nucleotide set.
As used herein, the term "first RNA pool" refers to all RNA contained in the nucleotides extracted from the sample, including, but not limited to, host rRNA, host tRNA, host mRNA, pathogen RNA. In some embodiments, the first RNA set refers to the set of remaining RNAs after the DNA therein has been experimentally removed on the basis of the first set of nucleotides.
As used herein, "second RNA pool" refers to a pool of RNA that has been further depleted of host rRNA based on the first RNA pool. In some embodiments, the second RNA pool includes, but is not limited to, host rRNA, host tRNA, host mRNA, pathogen RNA. In some embodiments, the second set of nucleotides includes a second set of RNAs.
As used herein, "magnetic bead" refers to a substance having a surface labeled with a functional group capable of undergoing an adsorption reaction with a nucleotide. In some embodiments, the amount of magnetic beads used is identified with a multiplier "x", or the use of a multiple indicates the volume of magnetic beads, indicating how many times the volume of magnetic beads were used relative to the original sample volume. In some embodiments, the volume of beads used is 1×100 μl=100 μl, e.g., the original volume of sample is 100 μl, 1-fold beads, or 1×purified. In some embodiments, to achieve the purification of pathogen RNA or detection of a pathogen of the application, a first magnetic bead, a second magnetic bead, and/or a third magnetic bead is introduced.
As used herein, "first magnetic beads" refers to magnetic beads used in obtaining a first set of nucleotides, then subjecting the first set of nucleotides to DNA elimination, and enriching the remaining first set of RNA in the system. In some embodiments, the first magnetic bead is used in an amount of 1.6 times to 2.0 times, preferably 1.8 times the sample volume. That is, if the first magnetic bead is used in an amount of 1.6Xto 2.0Xor 1.6 times to 2.0 times when the volume of the sample in which the first nucleotide set is located is 50. Mu.l, the first magnetic bead is used in a volume of 80 to 100. Mu.L. When the amount of the first magnetic beads is 1.8Xor 1.8 times, the volume of the first magnetic beads is 90. Mu.l.
As used herein, "second magnetic beads" refers to magnetic beads used to remove components of non-pathogenic RNA from a second set of nucleotides or a second set of RNA. In some embodiments, the second magnetic bead is used to remove residual DNA and residual RNA in the second set of nucleotides or the second set of RNAs except for pathogen RNA. In some embodiments, the second magnetic bead is used in an amount of 2.0 x-2.4 x or 2.0 x-2.4 times the sample volume. Preferably 2.2 x or 2.2 times. That is, if the volume of the sample in which the second nucleotide set or the second RNA set is located is 50. Mu.l, the volume of the second magnetic beads used is 100 to 120. Mu.L when the amount of the second magnetic beads used is 2.0X to 2.4X or 2.0X to 2.4X. When the amount of the second magnetic beads is 2.2×or 2.2 times, the volume of the second magnetic beads is 110 μl.
As used herein, "third magnetic bead" refers to a magnetic bead used to enrich for pathogen RNA in the second nucleotide set or the second RNA set. In some embodiments, the third magnetic bead is used in an amount of 2.0 x-2.4 x or 2.0 x-2.4 times the sample volume. Preferably 2.2 x or 2.2 times. That is, if the volume of the sample in which the third nucleotide set or the second RNA set is located is 50. Mu.l, the volume of the third bead used is 100 to 120. Mu.l when the amount of the third bead used is 2.0X to 2.4X or 2.0X to 2.4X. When the amount of the third magnetic beads used was 2.2X1 or 2.2 times, the volume of the third magnetic beads used was 110. Mu.l. Those skilled in the art who have the benefit of this disclosure will appreciate from this disclosure that the selection, adjustment, and tuning of specific parameters for analysis paths may be made in accordance with the differences of specific examples without departing from the spirit of the application.
As used herein, "number of species sequences" refers to the length of a specific sequence read for detecting a pathogen during detection of the pathogen, particularly during cDNA sequencing of the pathogen, with a greater number of species sequences indicating greater detection accuracy.
As used herein, "depth" refers to the average number of sequencing of bases, such as 30-fold, compared to a known reference sequence in pathogen detection, particularly in pathogen cDNA sequencing, meaning that each base is measured an average of 30 times. In the present application, the higher the depth, the higher the confidence that the species is detected.
In the context of "RPM (Micro) -ratio", RPM, as used herein, is a normalized number of sequences, representing the number of sequences per million sequences aligned to the genome of a target species; RPM (Micro) -ratio refers in the present application to detecting pathogen RPM/background RPM. In the present application, RPM (Micro) -ratio is used to make a threshold judgment for a specific species, the higher the value is the better.
As used herein, "coverage" refers to the proportion of sequences sequenced to the entire genome in pathogen detection, particularly in pathogen cDNA sequencing, wherein higher coverage values indicate more reliable results for pathogen detection.
The inventors found that viral loads are often low in clinical samples. The use of macrotranscriptome sequencing to detect infection with RNA virus may result in the presence of undetectable RNA virus read lengths or very small amounts of RNA virus read lengths, and therefore enrichment of RNA virus and its nucleic acid is necessary.
Common methods for enriching target nucleic acids are two methods, forward enrichment of mRNA with T repeat oligonucleotides and removal of ribosomal RNA (rRNA). At present, a lot of reagents for removing human rRNA in samples based on different methods exist on the market, but the number of finished reagents applied to pathogen detection is small, and the difficulty is how to successfully integrate the rRNA removal step into the library preparation process, so as to realize the library construction, sequencing and analysis inspection process.
RNA extraction is required for RNA virus macro-transcriptome analysis, and high purity of RNA is required for stringent removal of DNA contamination. The clinical specimens are extremely human in origin. Even after sample pretreatment, nucleic acid extraction and purification in high throughput sequencing wet experiments, there are still residues of host DNA to varying degrees that affect RNA virus detection.
The content of rRNA in clinical samples is high, and the experimental flow of gene detection generally does not remove rRNA, which together results in low detection rate of RNA viruses. The experimental procedure of connecting rRNA removal operation with sequencing library preparation and other steps has higher technical requirements.
When the macro-transcriptome data of clinical samples are analyzed, more than 99% of the macro-transcriptome data are often host sequences, and a large amount of rRNA data are also included in the obtained sortable microbial data, so that the data available for RNA virus identification are very few, and the sensitivity of RNA virus macro-transcriptome sequencing is very low. In order to increase the detection rate of RNA viruses, it is necessary to increase the sequencing amount, which leads to an increase in sequencing cost and is not beneficial to the clinical application of macro-transcriptomes.
In order to increase the detection rate of RNA viruses, the application provides a novel method for purifying RNA virus nucleotide. In some embodiments, the RNA viral nucleotide purification process is: eluting the extracted total nucleic acid of pathogenic microorganism in working solution containing DNase I, incubating to remove residual host DNA, and purifying and recovering viral RNA. Such treatment provides for complete removal of host nucleic acids prior to library construction. In some embodiments, the optimized nucleic acid purification process of the application can thoroughly remove host nucleic acids, enrich target nucleic acids in wet experimental steps, and superimpose the target nucleic acids on subsequent bioinformatic analysis, thereby improving the detection rate of RNA viruses.
Further, in some embodiments, the RNA viral nucleotide purification process is: removing human rRNA from total RNA, constructing a library for sequencing, wherein the process is to remove the human rRNA from the RNA, reversely enrich virus nucleic acid to obviously improve the proportion of effective RNA, then carrying out conventional double-stranded cDNA synthesis, constructing a library for sequencing based on a transposase method, and successfully detecting target pathogens after a specific letter generation analysis process. The results show that the number of reads for RNA virus detection is significantly improved compared to the non-optimized procedure.
FIG. 1 is a schematic flow chart after optimization for improving RNA virus detection according to one embodiment of the present application. As shown in FIG. 1, according to one embodiment of the present application, the method for increasing the detection rate of RNA viruses in clinical samples based on a sequencing method comprises the following specific steps:
1. collecting clinical patient samples
Sample of clinical patient: fresh samples or frozen samples at-80 ℃. The alveolar lavage fluid, sputum and the like need to be added with a liquefying agent, and the sample is subjected to a second step of treatment or frozen at-80 ℃ within 1 hour after being homogenized.
DNase digestion, total RNA extraction
Total RNA extraction using pathogen DNA & RNA co-extraction kit. In this example, the nucleic acid of the pathogen in the sample was directly extracted using the Semer Feishul technology (China) Limited viral genome DNA/RNA extraction kit. The present application is not limited to a kit for extracting RNA. In some embodiments, the RNA elution volume is ≡15. Mu.L, preferably 35-50. Mu.L.
3. Residual host DNA removal, RNA purification: in the present application, the DNA removal of the residual host is performed using the following system:
after mixing, the mixture is placed for 5 to 15 minutes at room temperature, and RNA is purified and recovered. The preferable adding amount of DNase is 5-20U, and the effective removal of residual DNA corresponding to clinical samples can be realized. In some embodiments, further purification of RNA is achieved by recovering the purified RNA using magnetic beads of Norflua RNA, and this recovery method is only one embodiment of the present application and is not intended to be limiting.
4. Host-derived rRNA removal
The probe method is used to remove human rRNA, including a hybridization capture step and a specific nuclease treatment step, and according to one embodiment of the present application, a Ribo-off rRNA removal kit from Norpran can be used to reverse enrich the target RNA. In some embodiments, the removal of rRNA can also be performed by CRISPR/Cas9 system, and after shearing rRNA, the viral RNA can be enriched by magnetic beads or the like. The present application is not limited to a method for removing rRNA. 0.1-1ug RNA was diluted to 11uL using nuclease-free water.
1) Hybridization of RNA sample with probe:
95℃，2min；95～22℃，0.1℃/sec；22℃5min；
2) Adding RNase H to eliminate rRNA of DNA-rRNA heterozygous strand:
storing at 37 deg.c for 15-30 min and at 4 deg.c.
3) Dnase I digestion: adding DNase I to digest DNA, where the DNA also includes the remaining cDNA strands and primers):
the reaction procedure: storing at 37 deg.c for 15-30 min and at 4 deg.c.
4) Pathogen RNA purification
The product obtained by the above reaction was purified with 110. Mu.L of RNA Clean XP magnetic beads to obtain a product solution. The product solution contains reverse enriched target RNA, and the method comprises the following steps:
5. general reverse transcription reaction: adding the template, the reverse transcription buffer and the random primer into a PCR tube, uniformly mixing, carrying out warm bath for 3-8 minutes at 50-68 ℃, and then incubating on ice for 1-3 minutes. Adding reverse transcriptase mix by instantaneous centrifugation, mixing, incubating for 10 min at 25-30 ℃, incubating for 15-30 min at 40-50 ℃, incubating for 5 seconds-5 min at 80 ℃ to inactivate the reaction, and cooling at 4 ℃.
Synthesis of cDNA second strand: 10 mu L-50 mu L of cDNA product synthesized by reverse transcription, adding two-chain synthesis reagent on ice, incubating for 10 min-30 min at 16-37 ℃, and cooling at 4-10 ℃.
7. Double-stranded cDNA purification: the product was purified using DNA Clean XP beads and eluted with 15 to 30. Mu.L of nuclease free water.
8. Library construction
Library construction was performed using transposon library construction kit (DNA Library Prep Kit for Illumina, novzan) and the purified library was verified by bioanalyzer according to the instructions, the library being between 300-700bp.
9. High throughput sequencing and bioinformatics analysis
Deep sequencing was performed using a beta Nextseq CN500 sequencer by SE75, SE150, PE150, etc., preferably SE75.
In some embodiments, the sample contains a large amount of host DNA and RNA, and the abundance of RNA viruses is low, accounting for a small fraction of the sample in the host DNA removal step. It will be appreciated by those skilled in the art that only a very small portion of the resulting sequencing data can be used for species identification due to interference of host DNA and RNA. In addition, the high concentration of extracted nucleic acid also affects the input amount of the subsequent DNase, and more importantly, the residual DNA in RNA can affect the removal of rRNA, consume a library substrate, even occupy a large amount of sequencing data, and finally affect the detection rate of RNA viruses. Thus, in some embodiments, the inventors have performed a nucleic acid extraction step, i.e., host nucleic acid removal, which includes, but is not limited to: 1. host nucleic acid dissociation+degradation; 2. direct digestion of host DNA; 3. DNA digestion is performed during extraction of total nucleic acids of the pathogen; 4. total nucleic acids of the pathogen are extracted and, after obtaining pathogen DNA and/or RNA, host DNA digestion is performed. According to one embodiment of the application, policy 4 is preferably employed.
In some embodiments, methods for removing rRNA in an animal total RNA sample include, but are not limited to:
(1) Poly (A) extraction method, mRNA in eukaryote has a Poly (A) tail structure, and rRNA can be indirectly removed by enriching mRNA of eukaryote with Poly (T);
(2) Combining the hybridization product with rRNA-DNA probes (labeled with biotin groups) by using magnetic beads with streptavidin groups, thereby removing rRNA from total RNA;
(3) rRNA in the double strand is hybridized by utilizing RNase H enzyme to specifically degrade rRNA-DNA probe.
The probe of the method (3) covers the whole rRNA sequences of two species of human/mouse, so that the ideal rRNA removal effect can be achieved, and the cost performance is high. In the present application, the rRNA in the sample is preferably removed by the method (3).
In some embodiments, the present application provides for reverse transcription of a first strand synthesis module using NEB, and a second strand synthesis module using applicants self-made second strand synthesis modules. It will be appreciated by those skilled in the art that there are a variety of commercially available kits for achieving reverse transcription of RNA. The present application is not limited to the reagents used in reverse transcription and the reverse transcription method.
In some embodiments, the library construction method of the present application comprises: the DNA library Prep Kit V2 for Illumina was used for fragmentation, and specific linkers were added to both ends of the fragmented DNA fragments.
In the present application, the method for quantifying, quality testing and obtaining a DNA library comprises: in the fragment selection, the magnetic beads with the volume which is 0.7 times of that of the purified product are firstly used for removing large fragments, and then the magnetic beads with the volume which is 0.15 times of that of the purified product are used for removing small fragments, so that the optimal library fragment length is finally obtained, and the average library length is about 325 bp.
In the present application, the sequencing method is not limited. The application provides a method for sequencing on-machine and analyzing off-machine data, which comprises the following steps: after the data passed through human sources, low quality and low complexity sequences were removed, the sequence pathogens were annotated with the Kraken rapid classification software and reported for possible pathogenic pathogens.
In some cases, clinical RNA samples are limited in acquisition, and virus quality control of cell culture can be synchronously selected to be doped into negative clinical samples for optimization of experimental procedures.
In the present application, to implement an embodiment of the present application, an apparatus is referred to including: a radix angelicae full-automatic nucleic acid extractor, a biosafety cabinet, an oscillating metal bath, a PCR instrument, a magnetic rack, qubit4.0 and the like.
In the present application, to implement the embodiments of the present application, the reagents involved include: nucleic acid extraction kits, decompaction reagents, ribo-off rRNA Depletion kit (Human/Mouse/Rat), reverse transcription and two-strand cDNA synthesis reagents, library construction reagents, AMPure XP purification magnetic beads, qubit detection reagents, sequencing chips, and the like.
The present application will be further illustrated by a specific example.
Example 1: flow screening for efficiently removing residual DNA of a sample:
by incorporating RNA references into negative clinical samples: human orthopneumovirus, influenza A virus, human respiratory virus 3, etc., at a concentration of 250-2000 copies/mL. And then host nucleic acid removal is carried out on the simulated sample, and a process capable of better removing residual host DNA is screened out.
Two experimental combinations are set as follows: the combination 1 is to absorb the extracted magnetic beads of nucleic acid, digest DNA in DNase I-containing solution; the pathogen nucleic acid extracted in combination 2 was added to a solution containing DNase I and then recovered using RNA purification beads.
The combined 1 flow: taking 200-400 mu L of sample, adding 265-530 mu L of virus binding buffer solution and 5-10 mu L of proteinase K, thoroughly mixing, incubating for 10 minutes at 56 ℃ in a metal bath, transferring into a deep hole plate added with a nucleic acid extraction reagent, adding DNase I digestion after a washing step, rinsing with 80% ethanol, and eluting target nucleic acid in non-ribozyme water.
The combined 2 flow: taking 200-400 mu L of sample, adding 265-530 mu L of virus binding buffer solution and 5-10 mu L of proteinase K, thoroughly mixing, incubating for 10 minutes at 56 ℃ in a metal bath, transferring into a deep hole plate added with a nucleic acid extraction reagent, transferring target nucleic acid into DNase I working solution, digesting for 10-15 minutes at room temperature, and adding RNA magnetic beads to recover RNA.
The experimental results are as follows:
table 1: the amount of residual DNA in RNA obtained from two combinations
Wherein, the positive control is a reference mark used for indicating the normal operation of the reaction system in the qPCR system.
Table 2: number of readings of reference
Wherein, the samples 1-3 are alveolar lavage liquid, 4-6 are cerebrospinal fluid, and 7 are positive controls. In this embodiment, the positive control is used to indicate the normal operation of the reaction system, and the application does not limit the pathogen species in the positive control, and can indicate that the reaction system for detecting the reference is normal.
quantitative purification of human housekeeping gene β -actin in nucleic acids by qPCR results: combination 2 removed residual DNA more thoroughly. FIG. 2 shows the proportion of human nucleotides in the sequencing result after removal of sample DNA according to one embodiment of the present application; FIG. 3 shows the proportion of microbial nucleotides in the sequencing result after removal of sample DNA according to one embodiment of the present application. The sequencing method analyzes the proportion of the human nucleotide in the sample, and compared with the combination 1, the combination 2 has less proportion of the human nucleotide, and the proportion is reduced by about 20 percent; at the same time the microbial nucleotide ratio is increased by 10%. Table 2 shows that the number of read lengths detected for the reference article is increased by a factor of 2 to 10 or more compared with the combination 1 for the combination 2.
To sum up: for RNA viruses, the method of the application is preferably combined with the method of 2, and the removal of residual DNA in clinical samples is carried out, namely DNase I is added into the extracted total nucleic acid of pathogens, RNA is recovered, and the preferred dosage of DNase I is 5-20U, so that the effective removal of DNA in corresponding clinical samples can be realized.
Example 2: effect of rRNA removal in Total RNA on RNA virus detection efficiency
The extracted viral RNA contains residual host, other pathogenic DNA and human RNA (rRNA) besides target nucleic acid, and if the part is not removed, the viral RNA can be reversely transcribed along with the target nucleic acid, participate in a library building process, finally occupy a sequencing space and influence the detection rate of RNA viruses; however, at the same time, the rRNA removal procedure also removes part of RNA viruses, which affects the capacity and detection of RNA viruses, so how the actual detection process removes human rRNA from the obtained total RNA, the extent of the removal, etc. affects the final detection result.
The purpose of this embodiment is: the effect of the rRNA removal procedure on increasing the viral detection rate was tested according to the nucleic acid purification procedure in example 1 (combination 1) using the incorporation of RNA virus reference into negative clinical samples. The preparation of the simulated sample and the copy number of the incorporated reference were identical to example 1. In some embodiments, rRNA removal may use a Ribo-off rRNA removal kit from Northenzan corporation. The results were as follows:
TABLE 3 comparison of rRNA ratios in sequencing results
TABLE 4 read length vs. read length for incorporated reference for the remove/do not remove rRNA procedure
Wherein, the control is a sample without rRNA removal. The above samples 8-9 are alveolar lavage fluid and 10-11 are cerebrospinal fluid.
The result shows that the negative sample doped with the reference sample is subjected to human rRNA removal treatment and rRNA non-removal treatment respectively, and then reverse transcription and library construction are carried out by using the same flow, and the detection reading length of the rRNA non-removal sample reference sample is on the unit order level as seen from the sequencing result, and the rRNA ratio in the sequencing result is reduced by 2-38% through the rRNA removal step, and the detection reading length of RNA viruses is improved by 2-84 times. FIG. 4 shows the proportion of human nucleotides in the sequencing result after rRNA removal from a sample according to one embodiment of the present application; FIG. 5 shows the proportion of human nucleotides in the sequencing results after removal of rRNA from a sample according to one embodiment of the present application. As shown in the figure, after human rRNA in the sample is removed, the proportion of human nucleotide is obviously reduced, and meanwhile, the proportion of microbial nucleotide is obviously improved.
In conclusion, the total RNA is subjected to reverse transcription and library establishment after rRNA is removed, and the effect is remarkable on reducing the proportion of rRNA in a sequencing result and improving the detection rate of pathogens.
Example 3: host rRNA removal reagent screening
And (3) researching manufacturers with good rRNA removal effect on the market, finally determining finished product kits of two different manufacturers based on a probe method, and comparing, wherein the detection effects of the two different manufacturers are compared in parallel by using a simulation sample due to the same principle. The experimental procedure was negative with reference to RNA viruses incorporated into cerebrospinal fluid and alveolar lavage fluid, human parainfluenza virus type 2, human orthopneumovirus and influenza a virus. The results are shown in the following table:
TABLE 5rRNA reagent vendor screening
The main difference between the kit 1 and the kit 2 is that: the reaction principle of the two is different, and the specific steps are as follows:
the reaction principle of the kit 1 is as follows: probe hybridization+streptavidin magnetic bead method. Namely, adopting a biotin-marked DNA probe to carry out liquid phase hybridization with rRNA with high abundance in total RNA to form an RNA-DNA probe complex; then, combining the RNA-DNA probe complex and the redundant probes in the liquid phase by using magnetic beads marked by streptavidin; separating magnetic beads from liquid phase, and obtaining the residual RNA in the liquid phase supernatant as target RNA after rRNA and redundant probes are removed.
However, the method has the disadvantages of high sample initiation, high rRNA residual quantity and complex operation, and all factors affect rRNA removal efficiency.
The reaction principle of the kit 2 is as follows: probe hybridization + rnase H digestion. Namely, adopting a specific DNA probe to carry out liquid phase hybridization with rRNA with high abundance in total RNA to form an RNA-DNA probe complex; RNase H targeted digestion of RNA single strand on RNA-DNA probe complex, while rRNA not hybridized with probe is not affected; the remaining probe is digested with DNsse, and the target nucleic acid such as undigested rRNA is purified and recovered.
Compared with the kit 1, the method has the advantages of low initial sample quantity, lower rRNA residual quantity, relatively simple operation, suitability for rRNA removal of trace samples, and better rRNA removal effect than the kit 1 in RNA pathogen nuclear detection.
To sum up: the rRNA removal kit of the kit 2 has good overall effect, the host nucleotide ratio is obviously reduced, and the pathogen detection reading length is obviously increased. In some embodiments, the rRNA removal kit of selection kit 2 accomplishes the detection of the clinical samples of the present application.
Example 4: clinical sample detection
Through the investigation of examples 1, 2 and 3, the experimental procedure of the present application was determined, and the following results are merely shown as the results of the optimal scheme and are not to be construed as limiting the scope of the application. Based on the constructed macro transcriptome experimental flow for improving the detection rate of RNA viruses, the clinical samples which are positive to the RNA viruses through sequencing are tested. The experimental procedure is as follows: 200 to 400. Mu.L of the procedure for removing residual host DNA of the above combination 2 was performed, and then human rRNA removal treatment was performed by the method of example 2, and conventional reverse transcription and library construction were performed using the recovered RNA, and sequencing analysis was performed, with the results shown in the following Table:
table 6 comparison of pathogen detection of two procedures (remaining alveolar lavage fluid)
Wherein, the control flow is a flow without removing DNA and rRNA in the sample. The optimization procedure is a procedure for removing DNA and rRNA in the sample. As shown in the results of Table 6, the number of the read lengths of the positive samples detected by viruses is increased by 1.2 to 29.3 times through the detection of the optimized flow, and the performance is greatly improved.
TABLE 7 off-the-shelf data analysis of different pathogens
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Table 7 shows the results of the on-press data analysis for different pathogens. As shown in Table 7, after the pathogen RNA is purified by using the optimization flow, the indexes such as seed sequence number, depth, RPM (Micro) -ratio, coverage and the like are all improved compared with the control flow; wherein the number of seed sequences can be increased to 30 times before optimization. Therefore, after the pathogen RNA is purified by using the optimization procedure, the detection accuracy and the reliability of the pathogen are better than those of the pathogen by using the control procedure. The optimized flow of the application is more conducive to the detection of trace RNA pathogens and can improve the detection sensitivity.
The above embodiments are provided for illustrating the present application and not for limiting the present application, and various changes and modifications may be made by one skilled in the relevant art without departing from the scope of the present application, therefore, all equivalent technical solutions shall fall within the scope of the present disclosure.
Claims (17)
1. A method of obtaining viral RNA comprising:
removing host rRNA in the first nucleotide set to obtain a second nucleotide set; wherein the first set of nucleotides includes host DNA, host rRNA, and pathogen RNA; the second set of nucleotides includes pathogen RNA; and
purifying pathogen RNA in the second set of nucleotides.
2. The method of claim 1, further comprising:
removing DNA in the first nucleotide set to obtain a first RNA set; the first RNA set includes: host rRNA and pathogen RNA;
enriching the first RNA set in the total nucleotides of the sample to obtain an enriched first RNA set;
removing host rRNA in the enriched first RNA set to obtain a second RNA set, wherein the second RNA set comprises pathogen RNA; and
purifying pathogen RNAs in the second RNA pool.
3. The method of claim 1, wherein the second set of nucleotides comprises a second set of RNAs.
4. The method of claim 2, wherein the method of enriching the first set of RNAs comprises: the first RNA collection is enriched using first magnetic beads that are used in an amount of 1.6-2.0 times the sample volume, preferably 1.8 times.
5. The method according to claim 1 or 2, further comprising:
adding to the sample an rRNA probe configured to specifically bind to the host rRNA in the first nucleotide set or the first RNA set without binding to pathogen RNA; and
removing complexes of the rRNA probes that bind to the host rRNA from the sample.
6. The method of claim 5, further comprising: RNase is added to the sample, which digests host rRNA bound to the rRNA probe, but does not digest pathogen RNA.
7. The method of claim 1 or 2, the method of purifying pathogen RNA comprising: enriching the residual DNA and the residual RNA except for pathogen RNA in the second nucleotide set or the second RNA set using a second magnetic bead; the second magnetic beads are used in an amount of 2.0 times to 2.4 times, preferably 2.2 times the sample volume.
8. The method of claim 7, wherein the method of enriching pathogen RNA in the sample comprises: the pathogen RNA is enriched using a third magnetic bead in an amount of 2.0-2.4 times the sample volume, preferably 2.2 times.
9. The method of claim 1, wherein the sample is alveolar lavage, cerebrospinal fluid, blood, urine, stool, a respiratory tract sample, hydrothorax, ascites, pericardial fluid, vomit, abscess tissue.
10. The method of claim 1, wherein the pathogen is one or more of the following RNA viruses: human orthopneumovirus, rhinovirus A, human metapneumovirus (hMPV), GBV-C, human respiratory virus 3, human parainfluenza virus type 2, influenza A virus, hepatitis A virus, rotavirus, measles virus, AIDS virus, encephalitis B virus, influenza B virus, rhinovirus, polio virus, coxsackie virus, dengue virus, rotavirus, SARS virus, MERS virus, ebola virus, marburg virus, phage, novel coronavirus (COVID-19).
11. A method of increasing the detection rate of RNA viruses in a sample, comprising:
extracting sample nucleotides to obtain a first nucleotide set; wherein the first set of nucleotides comprises: host DNA, host rRNA, and pathogen RNA;
purifying pathogenic RNA using the method of obtaining viral RNA according to any one of claims 1-10;
reverse transcribing the pathogen RNA to obtain double-stranded pathogen cDNA;
constructing the pathogen cDNA library;
sequencing the cDNA library.
12. The method of claim 11, further comprising: purifying the cDNA.
13. The method of claim 12, purifying the cDNA by electrophoresis, and/or adding a third magnetic bead; the amount of the third magnetic beads used is 2.0 times to 2.4 times, preferably 2.2 times, the sample volume.
14. The method of claim 11, wherein the cDNA library is 300-700bp in length of double-stranded pathogen cDNA.
15. A kit for purifying viral RNA from total nucleotides in a sample, comprising:
a dnase configured to remove DNA in the first set of nucleotides to obtain a first set of RNAs; the first RNA set includes: host rRNA and pathogen RNA; or it is configured to remove DNA from the second set of nucleotides or the second set of RNAs;
an rRNA probe configured to bind to host rRNA in the first nucleotide set or first RNA set, but not pathogen RNA;
rnase configured to remove host rRNA bound to rRNA probes without removing pathogen RNA;
RNA magnetic beads configured to enrich the first RNA pool and/or reverse enrich for pathogenic RNA after removal of host rRNA; the RNA magnetic beads comprise: the first magnetic beads and/or the second magnetic beads.
16. A kit for increasing the detection rate of RNA viruses in a sample, comprising:
a dnase configured to remove DNA in the first set of nucleotides to obtain a first set of RNAs; the first RNA set includes: host rRNA and pathogen RNA; or it is configured to remove DNA from the second set of nucleotides or the second set of RNAs;
an rRNA probe configured to bind to host rRNA in the first nucleotide set or first RNA set, but not pathogen RNA;
rnase configured to remove host rRNA bound to rRNA probes without removing pathogen RNA;
RNA magnetic beads configured to enrich the first RNA pool and/or reverse enrich for pathogenic RNA after removal of host rRNA; the RNA magnetic beads comprise: the first magnetic beads and/or the second magnetic beads.
17. Use of a method for obtaining viral RNA according to any one of claims 1 to 10, or a method for increasing the detection rate of RNA viruses in a sample according to claims 11 to 14, for detecting RNA pathogens in a sample.
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